ABSTRACT

H3K56 acetylation (H3K56Ac) was first identified in yeast and has recently been reported to play important roles in maintaining genomic stability, chromatin assembly, DNA replication, cell cycle progression and DNA repair. Although H3.1K56Ac has been relatively well studied, the function of H3.3K56Ac remains mostly unknown in mammals. In this study, we used H3.3K56Q and H3.3K56R mutants to study the possible function of H3.3K56 acetylation. The K-to-Q substitution mimics a constitutively acetylated lysine, while the K-to-R replacement mimics a constitutively unmodified lysine. We report that cell lines harbouring mutation of H3.3K56R exhibit increased cell death and dramatic morphology changes. Using a Tet-Off inducible system, we found an increased population of polyploid/aneuploid cells and decreased cell viability in H3.3K56R mutant cells. Consistent with these results, the H3.3K56R mutant had compromised H3.3 incorporation into several pericentric and centric heterochromatin regions we tested. Moreover, mass spectrometry analysis coupled with label-free quantification revealed that biological processes regulated by the H3.3-associating proteins, whose interaction with H3.3 was markedly increased by H3.3K56Q mutation but decreased by H3.3K56R mutation, include sister chromatid cohesion, mitotic nuclear division, and mitotic nuclear envelope disassembly. These results suggest that H3.3K56 acetylation is crucial for chromosome segregation and cell division in mammals.

摘要

H3K56 乙酰化 (H3K56Ac) 首次在酵母中发现，最近据报道在维持基因组稳定性、染色质组装、DNA 复制、细胞周期进程和 DNA 修复方面发挥重要作用。虽然 H3.1K56Ac 的研究相对较好，但 H3.3K56Ac 的功能在哺乳动物中大部分仍是未知的。在这项研究中，我们使用 H3.3K56Q 和 H3.3K56R 突变体来研究 H3.3K56 乙酰化的可能功能。K-to-Q 替代模拟组成型乙酰化赖氨酸，而 K-to-R 替代模拟组成型未修饰赖氨酸。我们报告含有 H3.3K56R 突变的细胞系表现出增加的细胞死亡和剧烈的形态变化。使用 Tet-Off 诱导系统，我们发现 H3.3K56R 突变细胞中多倍体/非整倍体细胞数量增加，细胞活力降低。与这些结果一致，H3.3K56R 突变体损害了 H3.3 并入我们测试的几个中心和中心异染色质区域。此外，质谱分析结合无标记定量显示，受 H3.3 相关蛋白调节的生物学过程包括姐妹染色单体，其与 H3.3 的相互作用因 H3.3K56Q 突变而显着增加，但因 H3.3K56R 突变而降低。凝聚力、有丝分裂核分裂和有丝分裂核膜分解。这些结果表明，H3.3K56 乙酰化对哺乳动物的染色体分离和细胞分裂至关重要。质谱分析结合无标记定量显示，受 H3.3 相关蛋白调节的生物学过程，其与 H3.3 的相互作用因 H3.3K56Q 突变而显着增加，但因 H3.3K56R 突变而降低，包括姐妹染色单体凝聚，有丝分裂核分裂和有丝分裂核膜分解。这些结果表明，H3.3K56 乙酰化对哺乳动物的染色体分离和细胞分裂至关重要。质谱分析结合无标记定量显示，受 H3.3 相关蛋白调节的生物学过程，其与 H3.3 的相互作用因 H3.3K56Q 突变而显着增加，但因 H3.3K56R 突变而降低，包括姐妹染色单体凝聚，有丝分裂核分裂和有丝分裂核膜分解。这些结果表明，H3.3K56 乙酰化对哺乳动物的染色体分离和细胞分裂至关重要。